Sub-nanomolar Detection of Prostate-Specific Membrane Antigen inSynthetic Urine by Synergistic, Dual-Ligand PhageKritika Mohan,§ Keith C. Donavan,§ Jessica A. Arter,§ Reginald M. Penner,*,§,† and Gregory A. Weiss*,§,‡

Departments of §Chemistry, ‡Molecular Biology & Biochemistry, and †Chemical Engineering & Materials Science, University ofCalifornia, Irvine, California 92697-2025, United States

*S Supporting Information

ABSTRACT: The sensitive detection of cancer biomarkers inurine could revolutionize cancer diagnosis and treatment. Suchdetectors must be inexpensive, easy to interpret, and sensitive. Thisreport describes a bioaffinity matrix of viruses integrated intoPEDOT films for electrochemical sensing of prostate-specificmembrane antigen (PSMA), a prostate cancer biomarker. Highsensitivity to PSMA resulted from synergistic action by twodifferent ligands to PSMA on the same phage particle. One ligandwas genetically encoded, and the secondary recognition ligand was chemically synthesized to wrap around the phage. The dualligands result in a bidentate binder with high-copy, dense ligand display for enhanced PSMA detection through a chelate-basedavidity effect. Biosensing with virus−PEDOT films provides a 100 pM limit of detection for PSMA in synthetic urine withoutrequiring enzymatic or other amplification.

■ INTRODUCTION

More-effective biosensors could address a critical need fordetecting cancer-associated biomarkers. An estimated 29 000men in the United States will succumb to prostate cancer in2013.1 Unfortunately, the lack of validated clinical diagnosticmarkers complicates efforts to develop tests for early prostatecancer detection. For example, a recent report concludes thatthe prostate-specific antigen (PSA) test used for prostate cancerdiagnostics is more harmful than beneficial.2 Despite thiscaveat, PSA remains an important biomarker for detectingrecurrent prostate cancer. However, early detection of thedisease could enable more-effective treatment and prognosis.3

Thus, issues addressable by bioanalytical chemistry include thedevelopment of more-sensitive measurements of proteinconcentration and then applying such measurements to identifyand validate more-effective biomarkers.Unlike PSA, prostate-specific membrane antigen (PSMA)

concentrations in biological fluids appear to offer a more-usefulmetric for prostate cancer diagnosis and prognosis.4 Forexample, elevated PSMA levels have been observed in prostatecancer patients’ urine.5 The PSMA concentration increasesfrom 0.25 nM to approximately 3.5 nM in prostate cancerpatients’ biological fluids, including urine.6 PSMA, a 750-residue, 90-kDa glycoprotein, is overexpressed on the surface oftumor cells as a non-covalent homodimer in >94.3 and >57.7%of primary and metastatic prostate cancers respectively.7,8

Elevated PSMA levels also correlate with the aggressiveness oftumor growth.9 Thus, PSMA offers an important biomarker forthe development of biosensor-based diagnostic devices. Thisreport describes the development of a biosensor capable ofdetecting clinically relevant concentrations of PSMA (<0.25nM) in synthetic urine.

In 2003, Petrenko and Vodyanoy demonstrated the use ofwhole virus particles as a bioaffinity matrix for biosensors.10,11

In an improved generation of biosensors, T7 virus particles witha peptide antigen from the West Nile virus on their surfaceshave been incorporated into conducting polymers by Cosnierand co-workers to allow detection of antibodies to the WestNile virus.12 This strategy can offer higher density ligands forbiomarker binding, as T7 phage have a high density of peptidesdisplayed on their surface. Improving biosensor sensitivitythrough increasing the density of ligands on the phage surfaceinspired in part the approach reported here.M13 bacteriophage, or more commonly “phage”, serve as

receptors for biosensors reported by our laboratories. Virusesthat infect only bacteria, the M13 bacteriophage have a readilycustomized protein coat, which can be tailored to bind tocancer biomarkers.13 The M13 viruses have ssDNA encapsu-lated by approximately 2700 copies of the major coat protein(P8) and five copies each of the four minor coat proteins.Manipulating the encapsulated DNA can provide peptides andproteins fused to the phage coat proteins, which are displayedon the phage surface.13 Combinatorial engineering of suchpolypeptides allows molecular evolution to obtain displayedligands with specific binding affinities and specificities.14,15

For direct electrical detection of biomarkers, M13 bacter-iophage have been incorporated into films of an electronicallyconductive polymer, poly-3,4-ethylenedioxythiophene(PEDOT).16−20 Synthesis of the biosensor film is accomplishedby electropolymerizing EDOT on the surface of a planar goldelectrode from a solution that contains virus particles. During

Received: March 19, 2013Published: April 24, 2013

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biosensor measurements, the electrochemical impedance of thevirus−PEDOT film increases upon exposure to the biomarker,providing a quantifiable readout for analyte binding.21

Modifications to the biosensing films could further improvethe device’s limit of detection (LOD) for translationalrelevance. In a previous report, our laboratories describedphage-incorporated into PEDOT nanowires, which resulted inbiosensors with a >66 nM LOD for PSMA in synthetic urine.22

Conventional phage display results in a low density ofgenetically encoded ligands displayed on the surface of thephage. Here, we focus on increasing the density of such ligands,as a strategy for more-sensitive measurements with highersignal-to-noise ratios. The concept of “phage wrapping” toimprove ligand density builds upon our previous reports ofwrapping the negatively charged phage surface with positivelycharged polymers to prevent nonspecific binding to thephage.23,24 The approach takes advantage of the presence ofnegatively charged residues, one Glu and two Asp, on the N-terminus of each P8. Since each phage includes 2700 copies ofP8, such carboxylate-bearing residues result in a high negativecharge on the outer surface of the virus particle.25 As reportedhere, additional ligands wrapped onto the phage surface due tothis electrostatic interaction lead to enhanced affinity andselectivity for PSMA.

■ RESULTS AND DISCUSSION

Genetically Encoded, Phage-Displayed Ligands Tar-geting PSMA. The two forms of PSMA, monomeric anddimeric, offer different targets for ligand binding; the dimericform is overexpressed by prostate cancer cells, and themonomeric form offers a closely matched negative control fornonspecificity, as a protein only found in healthy prostate cells.9

The relative binding affinities of two previously reported phage-displayed ligands,22 phage-1 and phage-2 (sequences andnomenclature in Table 1), for the PSMA isoforms were firstexamined by ELISA (Figure 1). Phage-2 binds with higheraffinity to the PSMA dimer than phage-1. Neither phage-displayed ligand binds with significant affinity to the PSMAmonomer. Thus, the peptide ligands selectively bind thedimeric form of PSMA. The specificity of phage-displayedligands for the dimeric PSMA is critical for potential clinicalapplications. Additional negative controls include phage-displayed peptides targeting the blocking agent (bovineserum albumin, BSA) and Stop-4 phage targeting PSMA; thelatter phage includes an analogous phagemid packaged intophage without ligands displayed on their surfaces. As expected,the negative controls failed to show any significant binding.The two PSMA ligands, 1 and 2, provide a starting point for

the development of biosensors. For translational relevance, theLOD of the resultant biosensor must be <0.25 nM, and a high-signal-to-noise ratio is essential for definitive diagnosis. Intheory, the affinity of the ligands for their target analyte shouldcorrelate with their usefulness in biosensor applications. For

example, a higher affinity ligand could enhance sensitivity forthe analyte. In a previous report, we described using phage-displayed homologue shotgun scanning to improve phage-displayed ligand affinity for PSMA by >100-fold.22 However,this approach requires extensive mutagenesis and selections.Nature applies evolution-guided affinity maturation but also

relies on another approach for more-rapid affinity maturation.The immune system, for example, applies the principle ofavidity to boost the apparent affinity of a weaker initial lead.During the initial immune response, the IgM protein presentsreceptors in a decavalent format, allowing weak initial bindersto attain higher apparent affinity through proximity- andchelate-based avidity.26 The large phage surface with repeatitivestructural motifs appears well-suited to this approach, and,indeed, avidity effects are often present during phage-basedselections and screens.27 This concept could provide ageneralized method for expedient improvement of ligandaffinity and biosensor sensitivity.

Cycloaddition To Generate the Secondary Recogni-tion Ligand. To exploit this avidity effect, phage wrapping wasused to boost ligand density, subsequent affinity, the resultantsensitivity, and the signal-to-noise ratio of phage-basedbiosensors. Each “wrapper” consists of two parts linkedtogether by the CuI-catalyzed azide−alkyne cycloaddition(“click”) reaction (Scheme 1).28 The first component, anoligolysine (K14) peptide, provides affinity to the phage surface.For the click reaction, an alkyne (4-pentynoic acid) wascoupled to the N-terminus of the K14 peptide. The secondcomponent of the wrapper is the peptide ligand to PSMA. Inprevious studies, the PSMA-binding peptides, 1 and 2,exhibited limited solubility in water. The peptides weretherefore synthesized as fusions to the solubilizing peptide

Table 1. PSMA Ligands, Sequence, and Nomenclature

Figure 1. Phage-based ELISA comparing ligand binding to monomericand dimeric forms of PSMA. This ELISA includes PSMA monomer;all other reported experiments with PSMA apply the cancer-relevantPSMA dimer. Stop-4 provides a negative control with helper phagepackaging the phagemid DNA. Throughout this report, error bars forELISA data represent standard error (n = 3). All experimental datapoints with the exception of the negative controls (n = 1) include sucherror bars, though often these are quite small.

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sequence K3 on their N-termini. For the click reaction, the N-termini of the peptide ligands were coupled to an azide (4-azidobutanoic acid).The two parts of each wrapper were chemically synthesized

using solid-phase peptide synthesis and purified by reverse-phase HPLC before the cycloaddition reaction. Here, clickchemistry offers a convergent synthesis, and the reaction takesplace at room temperature and in aqueous solution.28 Theresultant secondary recognition ligands thus provide anoligolysine half to wrap the phage (termed KCS for “lysine,chemically synthesized”) and a second component, the PSMAligand (1 or 2), to bind to the analyte (Figure 2). The products

formed by the click reaction (KCS-1 or KCS-2) werecharacterized by MALDI-TOF MS (Figure S1, SupportingInformation) and purified by reverse-phase HPLC to anestimated 90% purity.Phage Wrapping To Maximize Ligand Density.

Wrapping the phage with chemically synthesized PSMA ligandsdescribed above clearly enhances binding affinity to PSMA(Figure 3). The phage-displayed ligand (phage-2) was wrappedwith the secondary recognition ligands (KCS-1, KCS-2, or amixture of the two) to generate a phage surface displaying twoPSMA ligands. The wrapped phage were then assayed forbinding to the PSMA dimer. Negative controls, which resultedin no detectable binding, included the PSMA ligands targetingBSA and Stop-4 targeting PSMA. Phage-2 wrapped with KCS-2

exhibits ∼50 times higher affinity for PSMA than phage-2without the ligand wrapper (Figure 3A). Additional optimiza-tion examined the concentration of the wrapper (Figure S2). Asa result, the wrapper concentration can maximize the liganddensity on the phage surface. The effectiveness of wrapping forimproved binding affinity is dramatically demonstrated bycomparing the binding affinities of the helper phage (KO7)versus KO7 wrapped with KCS-2 (Figures 3B and S3). Lackinga displayed ligand, KO7 phage displays no significant binding toPSMA, but KO7 phage wrapped with KCS-2 binds withsignificant affinity to PSMA. Taken together, the resultsdemonstrate that the wrapping strategy achieves much higheraffinity for the target, and the wrapped ligands remainfunctional.

Primary and Secondary Recognition Ligands onPhage for Bidentate Binding. The arrangement and densityof the primary, genetically encoded ligand and the secondary,chemically synthesized ligand determine the affinity of the

Scheme 1. CuI-Catalyzed Azide−Alkyne CycloadditionReaction To Provide the Secondary Recognition Ligand,KCS-2

Figure 2. Schematic diagram of bidentate binding to PSMA bychemically synthesized (KCS-1, green) and genetically encoded(peptide 2, red) ligands to PSMA (PDB: 1Z8L). The former ligandwraps non-covalently onto the negatively charged P8 proteins foundon the phage surface due to conjugation with a positively charged K14peptide (blue). Simultaneous binding by the two ligands provideshigher apparent affinity to PSMA. The inset depicts a simplifiedversion of the schematic appearing in the subsequent figures shownhere.

Figure 3. Phage-based ELISAs demonstrating the effectiveness ofligand wrapping. (A) Phage with chemically and genetically encodedligands bind with much higher apparent affinity to the targeted PSMA.(B) The chemically synthesized KCS-2 wrapper converts helper phageKO7, lacking a genetically encoded PSMA ligand, into a high-affinitybinding partner to PSMA. The decrease in apparent binding affinity atthe highest phage concentration could be attributed to steric effects.(C) ELISA comparing different ligand wrappers. The wrappercombination of KCS-2 and KCS-1 indicates a 1:1 (w/w) ratio of ligandwrappers.

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wrapped phage for PSMA. For example, wrapping phage-2 withKCS-1 results in phage with an apparent 4-fold higher affinity forPSMA than phage-2 wrapped with KCS-2 (Figure 3C). Peptide1, however, has a much lower apparent affinity for PSMA thanpeptide 2, as shown in Figure 1. Thus, the increased bindingaffinity of phage-2 wrapped with KCS-1 suggests that the twoligands target different sites on the surface of PSMA.Furthermore, a 1:1 mixture of KCS-1 and KCS-2 wrapped onthe surface of phage-2 offers intermediate affinity between neatKCS-1 and neat KCS-2. The results demonstrate that phage-2wrapped with KCS-1 results in improved affinity due to abidentate binding interaction. Conversely, phage-2 wrappedwith KCS-2 fails to access this bidentate binding mode. Thus,the two ligands displayed on phage, for phage-2 wrapped withKCS-1, can result in a chelate-based avidity effect, whichenhances binding affinity beyond the gains achieved purely bymaximization of ligand density.In practice, chelate-based avidity effects can be challenging to

design, as geometry and sterics must be satisfied to allow bothligands to reach an optimal interaction with the receptor. Infragment-based drug discovery efforts, for example, develop-ment of linkers with appropriate configuration is a non-trivialproblem.29 Phage wrapping provides a more-expedient solutionto this problem. The second ligand, presented by a non-covalently bound wrapper, can equilibrate on the phage surfaceuntil finding a satisfactory geometry to allow simultaneousbinding for synergistic effect.Biosensing with Virus−PEDOT Films. Can these

wrapped virus particles be exploited to create virus−electrodebiosensors with a higher sensitivity for PSMA? To explore thisquestion, films of PEDOT were prepared on gold electrodes byelectropolymerization in the presence of phage-2 (Scheme 2).

The PEDOT films formed in solution during electropolyme-rization associate with the negatively charged perchlorate ionsfrom the electrolyte solution as it is deposited onto the goldelectrode.30 Polymerization of EDOT in the presence ofnegatively charged phage particles leads to incorporation ofvirus particles into the polymeric film as counterion dopantsdue to electrostatic interactions.21 The cyclic voltammogramacquired during electrodeposition of the virus−PEDOT

bioaffinity matrix indicates that the maximum current increaseswith every deposition cycle, consistent with the expectedincrease in the surface area of the film during growth (Figure4A). SEM imaging confirms incorporation of phage into thebioaffinity matrix; we observe both filament-like and less-extended features having dimensions consistent with phageintegrated as rope-like bundles into the polymer at variousangles to the film (Figure 4B,C). The KCS-1 wrapper was thenapplied in vitro simply by exposing the resultant phage-2 filmfor a short time to an aqueous solution of the wrapper. For thebiosensing measurements, only the highest affinity ligandcombination of phage-2 wrapped with KCS-1 was used, and itwas studied in comparison to phage-2 films.

EIS To Quantify PSMA Binding. As we have observed inour prior work,21 the electrochemical impedance of the virus−PEDOT film increases as PSMA selectively binds to the phage-displayed peptide ligands (Figure 4D). The real component ofthe impedance, R, in particular, increases upon PSMA binding.In previous work, we demonstrated that the increase in R, ΔR,normalized by the initial resistance, Ro (ΔR/Ro), can becorrelated with the concentration of a target molecule.21 Here,impedance data were acquired in phosphate-buffered fluoride(PBF)−Tween buffer, spanning a frequency range from 0.1 Hzto 1 MHz in an electrochemical cell with a Pt counter electrodeand virus−PEDOT film electroplated on a planar gold workingelectrode (Figure 4E). The films incorporating phage-2wrapped with KCS-1 provide higher sensitivity for PSMAdetection than films incorporating unwrapped phage-2 (Figure4F). For example, the relative impedance change, ΔR/Ro, ateach concentration of PSMA is 3-fold higher. The noise presentin this measurement (estimated as the standard deviation forfive impedance measurements) is unchanged, resulting in amuch higher signal-to-noise ratio for phage-2 wrapped withKCS-1 relative to unwrapped phage-2. A series of negativecontrols validate the data obtained. The PEDOT filmsincorporating Stop-4 phage, PEDOT films lacking PSMAbinding ligands, and PEDOT films incubated with KCS-1 resultin no significant binding to PSMA, as expected. The specificityof PSMA binding was investigated by using an alternativetarget, transferrin receptor (TfR), which has 54% sequencesimilarity to PSMA. No significant binding affinity to TfR wasobserved, as expected. This experiment illustrates the negligiblechange in impedance caused by the wrapper due to its smallsize.

Calculating the Hill Coefficient. The biosensing dataacquired for phage-2 and phage-2 wrapped with KCS-1 targetingPSMA follow a Langmurian adsorption model. The acquireddata were fit to the following Hill equation:

=YYK

[L][L]

n

n nmax

d

where Y = ΔR/Ro, [L] is the ligand concentration, and n is theHill coefficient.31 Consequently, the dissociation constant, Kd,and n were determined for phage-2 (Kd = 54 nM, n = 1.3, LOD= 6 nM) and phage-2 wrapped with KCS-1 (Kd = 33 nM, n =1.5, LOD = 3.1 nM). Here, the two LODs, defined as 3× overbackground signal, were calculated from line fits to the datashown in Figure 4F. The response obtained for phage-2wrapped with KCS-1 displays a much higher signal-to-noise ratiocompared to that obtained for films incorporating only phage-2.Such sensitivity can play a crucial role in low-concentrationanalyte detection. The n-values obtained are >1, indicating the

Scheme 2. Polymerization of EDOT in the Presence of (A)LiClO4 or (B) Phage-2, Followed by Wrapping with KCS-1(Green and Blue)

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presence of multiple binding sites and a cooperative bindingeffect. Phage-2 can access cooperative binding due to theavidity effect of multicopy phage-displayed ligands. Wrappingphage with additional ligands leads to a further increase incooperativity for phage-2 wrapped with KCS-1. Again, thesynergism of the two ligands leads to higher PSMA bindingaffinity for phage-2 wrapped with KCS-1.PSMA Detection in Synthetic Urine. To further

demonstrate the usefulness of the approach for potentialclinical applications, biosensing data were next acquired insynthetic urine. This complex solution includes water, nitricacid, urea, sodium sulfate, potassium chloride, sodiumdihydrogen phosphate, sodium chloride, ammonium chloride,and 10 other components; the resultant solution has a high saltconcentration (a calculated osmolality of 516.2 mOsm/kg and apH of 5.8).32,33 The solution provides a good model for theclinical challenge of identifying cancer biomarkers found inurine samples. Substituting synthetic urine for PBF, impedancemeasurements with virus−PEDOT films were acquired asbefore (Figure 5A). Having already established phage-2wrapped with KCS-1 as the most effective ligand combination

for detecting PSMA in terms of sensitivity, specificity, andsignal-to-noise ratio, our experiments focused on this ligandcombination for synthetic urine-based biosensing. In thepresence of PSMA, the impedance scans for virus−PEDOTfilms of phage-2 wrapped with KCS-1 follow similar trends inboth PBF and synthetic urine. However, the lower frequencyranges differ dramatically for the negative controls. Forexample, the negative control with Stop-4 phage in PBFdisplayed a much higher ΔR/Ro; this response at lowfrequencies was suppressed in synthetic urine. Thus, themeasurements in synthetic urine resulted in higher specificityfor the PSMA−ligand interaction.Turning next to the measurement of PSMA concentration,

the synthetic urine solution also appeared to enhancemeasurement sensitivity. The calibration curves in PBF andsynthetic urine were overlaid for comparison (Figure 5B, greenand purple, respectively). At high analyte concentrations,measurements in PBF and synthetic urine are superimposable.Both conditions reach saturation at high PSMA concentrations,hence the overlap in device response. At the lowest PSMAconcentrations, the higher ΔR/Ro response in synthetic urine

Figure 4. Biosensing with virus−PEDOT films. (A) Cyclic voltammogram for depositing virus−PEDOT films on gold electrodes. (B) SEM image ofa PEDOT film. (C) SEM image of a virus−PEDOT film prepared under the same conditions. (D) Relative change in resistance, ΔR/Ro, versusfrequency for phage-displayed ligands targeting PSMA. Data collected at 1 kHz (highlighted) were used for the analysis of PSMA binding. (E)Schematic diagram of the biosensing experiment. (F) ΔR/Ro of the film increases with the PSMA concentration. Throughout this report, error barsfor the biosensing data represent standard error (n = 5). Data were fit to the indicated lines using the Hill equation, resulting in an R2 value of >0.99.

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can be attributed to higher sensitivity and selectivity for PSMAunder the high-salt conditions. Phage-2 film wrapped with KCS-1 targeting PSMA in synthetic urine yields a 100 pMexperimentally observed LOD. Applying the calculation todetermine LOD described above yields a 10 pM LOD for thedetection of PSMA in synthetic urine. Furthermore, thissensitivity requires no signal or enzymatic amplification. Asbefore, no significant change in impedance was observed for thenegative controls.The high salt concentration in synthetic urine appears to

prevent nonspecific charge−charge interactions. Binding of thebiomarker to the virus−PEDOT film generates a positive ΔR/Ro, whereas a negligible ΔR/Ro for the negative controlsindicates a nonsignificant extent of nonspecific binding by theanalyte. The resultant specificity increase in synthetic urineboosts the apparent sensitivity of the device by decreasingbackground binding. This effect also enhances the sensitivity byunmasking a higher concentration of ligands for analytedetection, which would otherwise be occluded throughnonspecific binding. Thus, a dramatic improvement inspecificity and sensitivity can be obtained through thedecreased nonspecific interactions. The results suggest a generalstrategy for improving biosensor performance through focusingon decreased nonspecific binding.

■ CONCLUSIONThe 100 pM experimentally measured LOD obtained forphage-2 wrapped with KCS-1 is a significant improvement overour previous effort, which had a >66 nM LOD22 for PSMA.This detection sensitivity satisfies a key requirement for clinical

applications. The chelate-based avidity effect and highspecificity obtained due to the bidentate binding modedistinguish this work from our previous efforts and alsoaccount for the greatly improved signal-to-noise ratio and LODobtained without enzymatic amplification. Furthermore,inclusion of synthetic urine as an analyte solution expandsthe applicability of the technique. However, the reportedbiosensing films have not been optimized. Further improve-ments could be made to achieve an increased response, withoutamplification, for lower PSMA concentrations, as demonstratedby earlier experiments with similar biosensing films.34

In conclusion, wrapping phage with additional ligands canincrease the target affinity due to a chelate-based synergisticeffect. Phage-2 wrapped with KCS-1 binds to PSMA with Kd =33 nM and LOD = 3.1 nM in PBF, and a 100 pMexperimentally measured LOD in synthetic urine. In the future,the biosensors will be optimized for improved PSMA detection.

■ ASSOCIATED CONTENT*S Supporting InformationAdditional materials and methods. This material is available freeof charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authorgweiss@uci.edu; rmpenner@uci.eduNotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSR.M.P. acknowledges financial support of this work from theNational Science Foundation (CHE-1306928), and G.A.W.acknowledges support from the NAID (1 R43 AI074163) andthe NCI (R01 CA133592-01) of the National Institutes ofHealth.

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Figure 5. Detection of PSMA in synthetic urine using virus−PEDOTfilm biosensors. (A) ΔR/Ro versus frequency for the detection, insynthetic urine, of PSMA. (B) ΔR/Ro versus PSMA concentration.The inset expands the low PSMA concentration region. Data were fitto the indicated lines using the Hill equation, resulting in an R2 valueof >0.99.

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